Various devices are known for isolating a retentate containing a high molecular weight material, such as DNA or protein, through centrifugal ultrafiltration. The yields and amounts of retentate achieved using these techniques vary greatly due to the size, shape and position of filter membrane, the positions of outlets and/or the presence of ledges, corners or compartments in the devices.
Often these devices have associated limitations or drawbacks. For example, a device may be ineffective to prevent filtration of retentate to near dryness, or may have a design that hinders access to, or prevents complete pipette recovery of, the retentate due to chamber geometry, surface tension spreading, or the like. Also, a device may attain only a low yield or poor separation, or may require excessive centrifuge times. Additionally, a device may be poorly adapted for, or entirely incapable of, being prepared by or being used with robotic or other automated devices. Further, a technique or device may be uneconomic due, for example, to inefficient utilization of filter membrane area, and/or to manufacturing cost, and/or to requiring a long centrifuge time.
Therefore, a need exists for a centrifugal ultrafiltration device that can be dependably manufactured and used.
There is also a need for a separation technique that is rapid, effective and amenable to automated implementation.
There is also a need for improved processes for the manufacture or assembly of filtration or concentration vessels.
One or more of the foregoing ends are achieved in accordance with the present invention by providing a separation vessel having a conical region extending to a closed tip and a port in the wall of the conical region covered by a filter. The filter has a pore size and structure such that when centrifuged, fluid material such as solvent and solutes with a molecular weight below a threshold level passes through the filter and out the port. The conical region has a cone angle that causes retentate accumulating on the inner surface of the filter to slough down into the closed tip. Advantageously, the filter covers an area substantially larger than the port and is supported by the underlying wall so that a large filter area is actively used, and also resists clogging, thus allowing fast filtration. Moreover, the conical shape, which may extend from a cylindrical proximal or upper body portion, subtends a large reservoir of material in the vessel while allowing the receiving end to be sized for retaining a relatively small or minor fraction, e.g., below two percent, or even below a twentieth of one percent, of the total volume as retentate. Preferably, the filter is welded or fastened around its edges to the wall of the vessel by a process such as heat fusing or solvent welding, and covers a region extending from above the port at least down to the port. The vessel may have an upper flange allowing it to drop into a standard concentration tube so that the filtrate leaving the vessel is retained in the concentration tube and may itself be further processed, analyzed or transferred. In one embodiment, a deflectable member or portion of the vessel body operates as a pressure vent between the interior of the concentration tube and the interior of the separation vessel, allowing the vessel to be centrifuged while tightly capped. This feature also adapts a vessel to be overfilled and safely processed in a common tilted rotor assembly without spillage or blowout, thereby increasing the achievable single batch concentration ratio.
The separation vessel may be assembled by positioning a shaped filter sheet within the cone area of the vessel using a tool having one or more heated areas shaped to define bonding segments in peripheral regions of the filter at which the filter is to be fused with the body of the vessel. Preferably, the filter sheet has a trapezoidal shape which curls, when inserted into the vessel, to entirely cover the interior surface of a truncated-cone region of the vessel wall. The vessel may be formed with an alignment rib or other feature along its interior surface positioned to engage an edge of the filter sheet and thus to orient and align the sheet as it is curled against the curved inner wall of the vessel during insertion. A ledge may further be provided to catch the curled, aligned filter in position when fully inserted. Bonding of the filter to the wall is advantageously carried out by supporting the vessel in a heat sink while pressing a hot iron against the inner surface to fuse the filter backing membrane to the vessel wall.
The invention in another aspect provides a centrifugal concentrator having an alignment structure, such as a rib, which extends in a plane through the concentrator tube axis and serves to align a wedge-shaped membrane squarely along the axis during insertion of the membrane and assures that the filter edges are located away from ports of the vessel. The filter may extend substantially the full circumference, so that the well-aligned edges abut and seal precisely when pressed in along the axial direction with a tack welder, such as a conical tip and/or slotted insertion/heat sealing tool. The tool may also melt the vessel rib over the seated butt edges. A seating ledge provided in the vessel wall to engage the top edge of the filter further aids in orienting or positioning the truncated cone filter membrane, and stabilizes filter position during handling or assembly.
In yet another aspect the concentrator tube is configured to fit into and be supported by a filtrate collection tube, and the concentrator tube has a top sealing surface with a deflectable sealing lip that seals against the cap of the filtrate collection tube. The lip deflects in response to outside pressure within the capped collection tube, opening during centrifugation to allow venting via a bypass channel so pressure may vent from the collection tube to the concentrator tube, without leaking or blowing aerosols out to the centrifuge drum. This allows the concentrator tube to be overfilled, i.e., to be loaded into a fixed angle carrier at a higher fill level such that the fluid contents wet the cap, and yet to be processed without spillover or leakage, thus increasing the attainable concentration ratio and enhancing the speed and yield of the concentration process.
The invention also contemplates a separation vessel manufactured with a clamshell construction as part of a vessel array having the form of a strip or row of two or more vessels. In accordance with this aspect of the invention, a sheet of suitable polymer material is formed with a number n of identically-shaped troughs, each trough corresponding to one-half of the desired chamber shape, and including one or more ports formed in a conically sloping region thereof. A sheet of filter material is then placed over the multi-trough polymer sheet, and may optionally be pressed into the troughs and sealingly attached to cover the ports. Attachment is done by advancing one or more tools, such as a press mold or a hot wire die, which advantageously may be advanced in a direction perpendicular to the plane of the sheet, avoiding shear movement at the surface of the filter. A second symmetrically shaped filter-bearing polymer sheet is then laid on top to complete each of the vessel chambers, and the two polymer sheets are bonded together, for example by heat fusion, solvent or ultrasonic welding, or the like, to form a strip of n vessels. The geometry of the strip preferably conforms to the lattice spacing of a standard microtiter plate or multiwell receiving tray, e.g., forms a row of n or m vessels spaced to fit a standard nxc3x97m array or rack into which the strip itself is to be loaded for centrifuging. The basic row may also be manufactured to fit a into a portion of a single row or column of the matrix array, allowing multiple different sets of vessel strips to be loaded in the same array, which may, for example have dimensions knxc3x97jm, where k,j are integers.
Advantageously, since each separation vessel or chamber of the assembled array is centered around the tip of the conical region, the retentate resides in a defined and regular lattice position, and is accessible by a direct and unobstructed axial motion, thus making the vessel array adapted to robotic processing, pipette transferor assay, or operations with mechanized handling equipment.
The vessels of the present invention advantageously present a large surface area relative to the effective volume of the vessel, and operate at high rotational speeds while maintaining an open surface filter surface during operation. The filter is broadly supported against the adjacent vessel wall, providing a large flow area via interstitial space for filtrate passing out the ports, and free of internal structural encumbrances that might catch fluid and diminish its efficiency.
Assembly of the vessel and filter membrane is accomplished in a preferred aspect of the invention by insertion of a shaped heating tool to heat peripheral regions of the filter membrane as it lies positioned against the wall of the vessel. The filter may be a regenerated cellulose material on a porous polyethylene backing, such that the tool may contact the cellulosic material without sticking, and melt the backing to the vessel wall. The vessel and filter may be placed between a heat sink, and a press plate, with the heated member contacting the press plate to transfer a defined dosage of thermal energy with controlled thermal characteristics to the weld areas. A superheated rod and thimble embodiment employs a two-step heater advance to preheat and then weld the filter in place. The vessel or press plate may be provided with protrusions or partial ribs to automatically center the heat transfer tooling in the vessel and assure complete welding of the intended weld lines in areas to seal the filter over the ports and prevent ballooning of its central region.